Duffy et al. BMC Biology (2020) 18:112 https://doi.org/10.1186/s12915-020-00803-6 REVIEW Open Access Modified nucleic acids: replication, evolution, and next-generation therapeutics Karen Duffy†, Sebastian Arangundy-Franklin† and Philipp Holliger* Abstract Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs. Nucleic acids: natural and expanded functions afford completely new modalities for therapeutic inter- A heritable genetic system is a defining requirement for vention (e.g., direct protein expression or specific and life. The natural nucleic acids, DNA and RNA, are ex- programmable modulation of gene expression) that can- quisitely suited to store and propagate genetic informa- not be easily accessed by other biologics or small mol- tion with sufficient stability and fidelity to support large ecule drugs. However, several challenges have thus far genomes but also the flexibility to enable evolution. Nu- prevented nucleic acids from reaching their full potential cleic acids are unique among biopolymers in that both as medicines including poor chemical and biological accessible information and functional capacity coexist in stability and narrow chemical diversity. a single molecule. Thus, function can be evolved at the To address these limitations, DNA and RNA analogues molecular level yielding nucleic acid-based ligands and have been developed and evaluated for their ability to enzymes. In addition, nucleic acids fold through predict- serve as useful biomaterials or functional molecules, en- able and (to a large extent) programmable interactions, code genetic information, and support evolution. These are highly water-soluble, and can be easily denatured synthetic genetic polymers, broadly termed xeno nucleic and refolded. These advantages underpin their manifold acids (XNAs), exhibit modified backbones, sugars, or uses in biotechnology, diagnostics, therapeutics, nano- nucleobases, and even novel bases or base pairs [10, 11]. technology, material science, synthetic biology, and data While natural nucleic acids may also be modified to storage. modulate their function in vivo (e.g., post-synthetic epi- The last four decades have seen the rise of nucleic genetic modifications) [12–17], we constrain ourselves acids as therapeutics [1], primarily in the form of anti- here to a discussion of nucleic acid congeners not found sense oligonucleotides (ASOs) [2], small interfering in nature. We do, however, consider certain modifica- RNAs (siRNAs) [3], aptamers [4–6], microRNAs [7], tions that occur sporadically in natural oligonucleotides, mRNAs [8], and gene-editing guides [9]. Nucleic acids such as 2′OMe or C5 pyrimidine modifications, to be XNAs in the case of fully (or heavily) substituted oligo- * Correspondence: [email protected] nucleotides, the likes of which are not found in biology. †Karen Duffy and Sebastian Arangundy-Franklin contributed equally to this Several new and prominent XNA chemistries are shown work. in Fig. 1. One attractive feature of XNAs is their gener- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK ally improved chemical and biological stability [18–20]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Duffy et al. BMC Biology (2020) 18:112 Page 2 of 14 Fig. 1 (See legend on next page.) Duffy et al. BMC Biology (2020) 18:112 Page 3 of 14 (See figure on previous page.) Fig. 1 The chemical diversity of XNAs. XNAs are often categorized by the component of the nucleotide (sugar, backbone, or base) carrying a modification. Shown here are XNAs discussed in this review, including both those of medical and historical relevance as well as several newly described chemistries. 2′F, 2′-fluoro; 2′OMe, 2′-O-methyl; LNA, locked nucleic acid; FANA, 2′-fluoro arabinose nucleic acid; HNA, hexitol nucleic acid; 2′MOE, 2′-O-methoxyethyl; ribuloNA, (1′-3′)-β-L-ribulo nucleic acid; TNA, α-L-threose nucleic acid; tPhoNA, 3′-2′ phosphonomethyl-threosyl nucleic acid; dXNA, 2′-deoxyxylonucleic acid; PS, phosphorothioate; phNA, alkyl phosphonate nucleic acid; PNA, peptide nucleic acid Decoration with diverse chemical substituents (e.g., demonstration of genetic function in an uncharged back- hydrophobic groups) can also yield improved properties bone chemistry for the first time as well as in vitro evo- and functionalities such as new structural motifs and en- lution of phNA aptamers [29]. hanced target binding [21–24]. While a wide range of As certain individual modifications become easier to XNAs have been synthesized at the chemical level, synthesize and their unique properties better under- representing a rich palette to draw from, their functional stood, combining two or more of these known modifica- potential and usage is only just beginning to be probed. tions to sugars, backbones, and bases will further push Here, we explore recent discoveries in the enzymatic the boundaries of chemical diversity in XNAs [30–35]. synthesis and functional exploration of XNAs with a The development of ever more diverse chemistries view towards therapeutic applications. raises the question: how much of what we know about natural nucleic acids translates to XNA? To what extent Expanding the chemistry of nucleic acids can we borrow design strategies from natural nucleic Advances in nucleic acid chemistry have enabled the de- acids in order to rapidly develop useful new XNA chem- velopment of XNAs with improved base-pairing stability istries? Some design rules, such as the ability of certain over natural nucleic acids as well as enhanced activity in sequence motifs to form G-quadruplexes, have been the context of living tissues, leading to several FDA ap- shown to translate to TNA [36] and FANA [37]. How- proved nucleic acid therapeutics [25]. Research con- ever, the generality of such parallels remains to be explored. tinues apace to identify novel chemistries with increased potency, bioavailability, stability, decreased toxicity, and Templated synthesis of non-natural nucleic acids minimal off-target effects. The phosphoramidite approach to solid-phase DNA syn- An interesting development in this context is the dis- thesis [38, 39] developed in the early 1980s led to the main- covery that mixed backbone chemistries can display stream use of synthetic DNA in a myriad of applications. novel emergent properties. For example, Krishnamurthy Similarly, improvements in the synthesis of XNAs can be and colleagues probed the ability of XNA-RNA chimeric expected to increase their usage and unlock new applica- oligonucleotides to hybridize with their natural nucleic tions in the coming decades. XNA synthesis can be broadly acid counterparts and found that the otherwise non- divided into enzymatic and non-enzymatic approaches. pairing ribuloNA can be interspersed with RNA or DNA Some widely used XNAs (e.g., 2′OMe, LNA, PS, 2′MOE) building blocks to yield oligonucleotides with compar- can be chemically synthesized, although yields of even the able or higher duplex thermal stability than natural sys- most synthetically accessible XNAs are limited to around tems and tuneable base-pairing properties [26]. The 150 bp [40]. For other XNA chemistries, no reliable solid- authors suggest that such a system could be used to phase synthetic route is known [41]. Furthermore, solid- mimic natural base-pairing rules with a reduced number phase synthesis depends on explicit knowledge of the se- of bases, storing information in the sugar in place of the quence being synthesized. In contrast, templated synthesis GC base pair. enables general information transfer and is essential for the Similarly, the recent discovery that smaller “skinny” evolution of functional oligonucleotides. (pyrimidine-like) and larger “fat” (purine-like) base pairs can form stable helices challenges conventional assump- Non-enzymatic templated synthesis tions of nucleic acid structure [27]. While the novel base Non-enzymatic templated synthesis